ABSTRACT ?-Crystallin is a complex macromolecule that accounts for nearly 40% of the adult lens proteins. The chaperone-like activity of ?-crystallin, is implicated as a key component in the maintenance of lens transparency by suppression of crystallin aggregation. In vitro studies of ?-crystallin have shown that chaperone activity is increased when ?-crystallin is subjected to heat (48-60oC) and then brought back to 25- 37oC. Similarly, ?-crystallin subjected to urea-induced unfolding and refolding also displays increased chaperone activity. Understanding the molecular organization and properties of crystallin subunits in activated chaperones would help answer questions on how ?-crystallin chaperone-like activity might be harnassed and manipulated for the development of protein-based therapeutics. In our studies of the role of subunit interactions in chaperone activity, a recombinant ?B-crystallin expressed after deleting the 54-61 sequence (resulting in a protein designated as ?B?54-61) was found to form ~ 40 % smaller oligomer than the wild-type protein oligomer but to show a 10-fold increase in chaperone activity. The Specific Aims of this project will uncover the molecular changes that account for the increased chaperone activity in heat- and urea-treated ?-crystallin and deletion mutant of ?B-crystallin. Specific Aim 1: a) Determine the mechanism of ?B-crystallin chaperone activation after deletion of the 54-61 sequence and b) determine the biological implications of enhanced chaperone activity in cell culture system. Specific Aim 2: a) Determine the functional units and mechanism of activation in ?-crystallin chaperone after thermal stress and urea-induced unfolding and refolding and b) investigate the cytoprotective effect of heat- and urea-activated crystallins. Novel cross-linker(s) will be used to gain fresh insights into the ?cryptic? chaperone sites getting exposed in the activated crystallins. The studies will also make use of site-directed mutagenesis, mass spectrometric analysis and biophysical techniques to uncover the molecular changes at the secondary and tertiary structure levels and to delineate the quaternary organization of the subunits in the oligomers showing increased chaperone activity. To see whether the activated ?-crystallins can be exploited to protect cells from oxidative injury, the effects of stress-inducing agents such as H2O2, staurosporine or etoposide will be investigated in Cos-7, HeLa, HEK293 and ARPE-19 cells. Further, the ability of activated chaperones to suppress aggregation of mutant proteins (?AG98R) as well as fibril-forming ?-amyloid will be investigated both in vitro and ex-vivo. The long-term goals of the studies are to understand the structure?function relationship of activated ?-crystallin and develop crystallin proteins that have therapeutic value in protein conformational diseases.